The present invention relates to compositions, structures formed by the compositions, processes of forming the compositions and the structures, and detection processes relying upon the compositions and structures. More specifically, the present invention relates to fluorous-modified nucleoside, nucleotide, and oligonucleotide arrays, fluorous-modified nucleoside, nucleotide, and oligonucleotide array preparation and detection processes, and fluorous-modified nucleoside, nucleotide, and oligonucleotide array compositions.
Nucleosides, nucleotides, and oligonucleotides are amongst the most important classes of chemical compounds in modern molecular biology, medicine, and drug discovery. Their importance is not only exemplified in DNA and RNA, which are both oligonucleotides, but also in nucleotides such as adenosine triphosphate (ATP), which is the primarily molecule used for cellular energy transfer, and in nucleoside analogs such acyclovir, an important antiviral drug. Additionally, these compounds, collectively referred to herein as “nucleotides,” are of interest in many biological processes currently being explored for use in the diagnosis and treatment of various disease states. Accordingly, nucleotides are often used as substrates for the investigation of biomolecule interactions, small molecule inhibition of such interactions, and in the elucidation of biochemical pathways.
Assays and screens utilizing nucleotides as substrates or probes have become an integral part of the drug discovery and development process from target identification and validation through hit optimization and lead optimization. In addition, nucleotide-based diagnostics and therapeutics are increasing with nucleotide-based assays and screens playing an important role in drug development and clinical applications. The development of assays and screens using nucleotides as probes that provide high quality and high throughput is a continuing area of interest to the life sciences industry. However, there are many challenges to using nucleotides as molecular probes. One such challenge includes the unique chemical and physical properties of nucleotides, as compared to peptides, proteins, and carbohydrates, which are the other major classes of biologically relevant compounds.
There are a number of methods used currently for nucleotide-based assays. The standard methods are solution-phase-based methods using microtiter plates containing a number of wells generally ranging from 96 to 1536. The test solution and compounds along with a labeled nucleotide probe are added to each well. The label is generally a fluorogenic or chemiluminescent label which is necessary for detection. After the reaction is complete, the plate is scanned and those reactions which were positive can be distinguished from those that are negative through the fluorescent or chemiluminescent label. Microtiter-plate-based solution phase assays can often be conducted with very high throughput resulting in large data sets that are highly data dense. The primary shortcoming of these types of assays; however, is that the data is generally not of high quality.
Low data quality is generally due to the use of the fluorescent or chemiluminescent detection label. The label itself can oftentimes interfere with the native activity or selectivity of the nucleotide of interest. This results in a high rate of false positives and negatives. In addition, when adding test compounds that are potential drug candidates, fluorescence and chemiluminescence detection can be compromised either by autofluorescence or by interference from the test compound. It is common for 10% fluorescence interference rates to be reported when screening a library of compounds using assays of this type.
In addition, the information gathered by using fluorescent or chemiluminescent detection does not provide any structural information. Depending on the design of the assay, information such as the degree of change imparted on the nucleotide probe or the exact location of that change is lost. For example, in a ribose nucleic acid (RNA) screen, a variety of putative nucleotide substrates may be examined using a fluorogenic label. Cleavage of the RNA at the designed nucleotide location results in a positive signal. However, if hydrolytic activity were to take place concomitantly at other sites within the oligonucleotide backbone, a false positive signal would result providing an erroneous result. Alternatively, if two putative sites of cleavage were available on the nucleotide, a positive signal would indicate that some cleavage took place, but would not distinguish at which location. So, while the solution-phase-based assays can often be high throughput, they suffer from producing low-quality data which often requires extensive re-testing by other methods in order to confirm the results or to obtain additional information.
Another limitation of many solution phase assay systems is the inability to enrich the test sample for the analytes of interests. Analyte enrichment results in greater signal to noise, higher sensitivity, and lower issues with signal interference. This has led to the development of various labels designed to facilitate analyte enrichment in addition to detection, such as biotin-streptavidin, polyhistidine (His) tags, or solid phase tags. In the case of biotin or His tag labeled nucleotides, the labeled analytes can be enriched using the appropriate affinity support; streptavidin in the case of biotin and a nickel or cobalt based resin for His tags. However, many affinity based enrichment methods are limited by non-specific binding and lack of bioorthogonality, which gives rise to false signals requiring substantial follow-up experiments. For example, when using biotin-streptavidin based assays false signals can occur due to a number of factors including endogenous biotinylated molecules, biotin complexation with other proteins such as carboxylases, or streptavidin binding with other ligands besides biotin.
In contrast to solution-phase assays, the nucleotide probes in solid-phase assays are immobilized, usually through covalent bonding to a surface, for example, the bottom of a well within a microtiter plate or a three-dimensional solid such as a bead. The test solution containing the protein or enzyme, test compound, and/or other reaction components is added. Once the reaction is complete, the solution is removed and the well is washed to remove all other components leaving only the immobilized probe nucleotide. If the nucleotide is labeled, the fluorescence or chemiluminescent detection can be used as in the solution-phase assay.
The major advantage of the solid-phase method is that auto-fluorescent or fluorescence-interfering materials can be washed away reducing the number of false readouts. This does not, however, overcome limitations due to the presence of the label itself which may interfere with the action of the protein, enzyme, or other interacting entity nor does it provide additional structural information missing from the solution-phase assay.
Both solid-phase and solution phase assays may also use other detection methods. One known method is a radiometric method where a radioactive-isotope-labeled atom is incorporated into the nucleotide either before or after the desired reaction. Popular radioisotopes include 14C, 32P, 25S and 152I. Radiometric methods are extremely sensitive and can oftentimes be quite specific. The major disadvantages are the special care and precautions necessary when using radioactive materials and the cost of the isotopes which can preclude use in early screening efforts. In addition radiometric detection provides no structural information such as the location of the radioisotope incorporation within the nucleotide nor does it provide a reliable measure of degree of radioisotope incorporation.
Another known detection method is a coupled assay which utilizes a second reaction in order to introduce a fluorescent or chemiluminescent label. In a coupled assay, label-free nucleotides can be used as the probe thereby avoiding any questions as to the effect of the label on activity. After the desired reaction is complete, a secondary reaction, generally using a labeled specific antibody, is conducted. The specific antibody only binds to the transformation in question, thereby providing the fluorescent signal. A solid-phase assay using a coupled antibody reaction therefore overcomes the problem of the labeled nucleotide probe and the interference issues often associated with solution-phase assays. Once again; however, it does not provide information-rich data.
In addition, the coupled assay can require introduction of appropriate surface chemistry in order to immobilize the probes. A large number of different surface chemistries have been introduced for probe immobilization including affinity-based such as biotin-streptavidin, and covalent bonding based as in maleimide, Diels-Alder, click chemistry, etc. Non-specific binding is also often a problem with these methods causing binding of other molecules besides the probe molecule to the surface compromising signal to noise ratio. For example, if the antibody used in the coupled assay non-specifically binds to the surface, then a false positive signal will be received. Also, the coupled assay is often dependent on the quality of the antibody which is a well-known problem in biological research as variability in antibody activity and selectivity, both inter- and intra-batch, introduces reproducibility and reliability issues in the data generated. In the absence of a highly selective antibody, the assay will once again result in a high number of false readouts. This lack of specific antibodies is in many areas considered to be one of the biggest shortcomings within the field.
Despite these drawbacks in information quality and robustness, solid-phase assays have found widespread use within the life sciences industry, primarily due to the high throughput that can be achieved, the epitome of which is the microarray where thousands of probes can be applied to a small surface and interrogated at once. Microarrays, including nucleotide microarrays, have been demonstrated extensively. Beyond the high throughput, the miniaturized format of microarrays allows minimal use of probe and reagents thereby reducing the overall cost per probe relative to microtiter-plate formats. The microarray format does not overcome the detection problems; however, of other assay formats.
An additional limitation of many of the above described methods are the expense of the fluorescent or chemiluminescent labels which add significant cost to utilizing these assays in the screening of large compound collections.
Other label-free detection methods include optical methods, such as surface plasmon resonance (SPR). SPR provides no structural information but is a sensitive method by which to observe changes in probes or to detect binding events. Even so, there are some transformations, such as phosphorylation, which SPR cannot reliably detect.
In order to address data quality and cost issues, researchers have turned primarily to mass spectrometry (MS) as a label-free detection method. Mass spectrometry is particularly well-suited for binding and activity assays since it can not only detect both starting probes and products, but also determine the degree of change on the probe and the location of the change. MS-based assays therefore provide informational richness that other detection methods do not afford leading to more robust data free from many of the issues faced using light-based detection methods. For example, a recent report of a high throughput screen found that using fluorescence detection 92% of positive signals were false and that converting the screen to MS detection reduced that false signal rate to the single digit range.
While providing high quality data, MS generally suffers from being only low to medium throughput. This is due primarily to the necessary sample preparation and purification in order to remove impurities and other unwanted materials from the sample which can adversely affect sensitivity and detection. A favored method to desalt a sample prior to MS is to use a solution-phase assay and liquid chromatography and MS (LC/MS). The chromatographic separation purifies the probe of interest so that it can then be analyzed by MS. This method; however, is very low throughput with each sample requiring a minute or more of time to analyze. Other methods, such as multiplexed (MUX) electrospray with parallel LC systems, have reduced the analysis time to as short as 30 seconds/sample, but still are orders of magnitude behind high-throughput assay methods. Another recent system includes a microfluidics-based desalting system coupled with MS detection. This system has been reported to process samples at a rate of one every 5-7 seconds which is significantly higher than other MS-based methods but still falls far short of fluorescence or radiometric-based systems.
Examples of assays which utilize a nucleotide with MS detection are kinase or phosphatase assays using adenosine triphosphate (ATP). Kinases transfer a phosphate from ATP to a protein therefore transforming ATP to ADP, adenosine diphosphate. The enzymatic activity can then be easily determined by the ATP:ADP ratio as determined by MS detection. Another example of MS detection of a nucleotide in a pharmaceutically relevant assay is the use of S-adenosyl methionine (SAM) in protein and peptide methylation. After methyl transfer, SAM is transformed into S-adenosyl homocysteine (SAH). Once again the level of enzymatic activity can be determined by measuring the SAM:SAH ratio. A significant advantage in these MS-based assays is that both the starting material and product levels can be measured which is generally not possible with other detection methods. Measuring only starting material consumption or product formation, but not both, can often lead to erroneous results including false positives and negatives should other processes than the desired one be occurring resulting in loss of starting material or accumulation of product.
In recent years there has been considerable effort to increase the throughput of MS analysis through the use of matrix-assisted laser desorption/ionization MS (MALDI-MS) or laser desorption/ionization MS (LDI-MS) which does not employ an external matrix. MALDI and LDI methods promise higher throughput than LC/MS methods and are particularly well-suited for the detection of various types of enzyme substrates, particularly peptides.
Unfortunately, the MALDI-MS detection of oligonucleotides is not as straightforward as that of peptides, primarily due to the presence of the phosphodiester linkage in the oligonucleotide backbone. The anionic phosphates render oligonucleotides highly prone to retaining salts and other contaminants thereby suppressing ionization, forming multiple mass species, and inducing gas phase fragmentation, all of which lead to poor sensitivity and reproducibility. This has led to a plethora of different desalting and matrix formulas and protocols designed to overcome the salt issue. Examples include the addition of co-matrices such as spermine, ammonium salts, or other polyamines, pre-treatment with anion exchange resins, or development of exotic matrix compounds. This is in direct contrast to MALDI-MS analysis of peptide or carbohydrate substrates which generally ionize readily through the addition of a simple single component matrix. The challenges in using MALDI-MS for high throughput screening with oligonucleotide probes is highlighted by the lack of such assay platforms relative to peptide based assays.
MALDI or LDI-MS based arrays have been developed in an attempt to combine the high-throughput of the microarray format with the high quality data of MS detection. A major difficulty in combining microarrays with MS detection is that methods for immobilization of nucleotide probes generally utilize covalent bonding formed by reaction of a reactive group on the nucleotide and a reactive group on the surface. The covalent bond formation allows the probes to be immobilized in a specific orientation and through a specific location on the nucleotide. Most covalently bound probes, however, cannot be ionized from the surface once the covalent bond is formed making it incompatible with direct MS detection. Non-covalent immobilization methods have also been used to form microarrays but generally lack specific display orientation which can affect substrate activity. There are, however, methods which can combine a microarray format with MS detection using non-covalent immobilization with specific display orientation.
A primary example of this would be self-assembled monolayer desorption/ionization MS (SAMDI-MS) where enzyme substrates are immobilized through the formation of alkanethiol monolayers on a gold surface. The substrates can then be reacted with the test solution of interest. A chemical matrix is then added to induce ionization and the probes are then analyzed by matrix-assisted laser desorption/ionization (MALDI-MS). SAMDI-MS then combines the high throughput of microarrays with the high quality data of MS detection.
The method, however, suffers from a number of deficiencies. First, results can be highly variable and dependent on the choice and application of the matrix. The optimal matrix can vary depending on the monolayer employed and the nature of the probes. Application of the matrix also needs to be precisely controlled in order to avoid inconsistencies and “patches” across the array resulting in areas of poor signal. In addition, the monolayers themselves may not be robust and are subject to degradation at elevated temperatures and upon exposure to UV light. These issues lead to questions regarding long-term storage and scalability, both critical items for commercialization.
Another limitation of SAMDI presents itself in the specific case of oligonucleotide probes. The SAMDI methods which were effective for peptide and carbohydrate immobilization and laser ionization proved to be completely ineffective when using oligonucleotide probes (Tsubery and Mrksich, Langmuir, 2008, 24, 5443). Formation of the self-assembled monolayer using thiol modified oligonucleotides occurred readily but ionization and detection by MS of the immobilized oligonucleotides using a variety of matrices proved completely unsuccessful. In order to utilize SAMDI for oligonucleotide probes it was necessary to first form a monolayer of biotin which was then reacted with streptavidin. The resultant streptavidin modified surface could then immobilize biotin modified oligonucleotides which could then be ionized from the surface after matrix addition. The use of biotin-streptavidin affinity, however, introduces its own issues including non-specific binding to both components. The unexpected difficulties in using oligonucleotide probes along with the cumbersome workaround has limited the utility of SAMDI in oligonucleotide applications. The authors noted that it was unclear why their previous protocols which worked effectively for other molecular classes (peptides, proteins, and carbohydrates) failed to do so for oligonucleotides. The work highlights the difficulty and unpredictability in applying prior MALDI-MS methods to oligonucleotides.
There have recently been efforts at avoiding some of the issues described. One method is to utilize fluorous partitioning where a highly fluorinated surface is used to immobilize perfluorocarbon modified substrates. Fluorous partitioning takes advantage of the fact that fluorous phases represent a third distinct phase from organic and aqueous phases and that fluorous tagged molecules partition preferentially and selectively into a fluorous phase. As such, the fluorous phase and fluorous tags are both hydrophobic and lipophobic. Since fluorous molecules are not biologically endogenous the use of fluorous partitioning is completely biorthogonal which is a highly desirable characteristic when conducting analyses from complex biological mixtures.
Fluorous partitioning has primarily been used as a separation technology in the synthesis and purification of various molecular classes including small molecules, peptides, carbohydrates, and oligonucleotides. In these applications, a temporary fluorous tag is attached to a molecule which can then undergo a series of synthetic reactions. The fluorous tagged molecule can then be separated from excess reagents and other non-tagged components through fluorous partitioning with either a liquid or solid fluorous phase. After removal from the fluorous phase, the fluorous tag can then be cleaved from the molecule providing the purified target molecule free from the fluorous tag. Alternatively, the desired target molecule can be non-tagged while reagents of undesired compounds can be fluorous tagged and a fluorous partition-based separation can be used to separate the target compound from the undesired compounds.
Fluorous partitioning has also been used for immobilization using a fluorous planar solid surface as the fluorous phase. Fluorous immobilization combines aspects of non-covalent and covalent immobilization. The probes are immobilized non-covalently through fluorous partitioning but in a specific display orientation through a specific end of the molecule; a characteristic usually reserved only for covalent bonding motifs. MS detection can then be conducted either through the use of a nano-structure initiated MS (NIMS) or directly off the surface, in some cases by laser ablation without the need of matrix. These non-matrix methods also have the advantage of being highly robust systems that require a minimum of special storage conditions.
To date fluorous partitioning has been used for the immobilization of fluorous tagged small molecules, peptides, and carbohydrates. Fluorous immobilization with direct MS detection has been reported for carbohydrates, but no reports of nucleoside, nucleotide or oligonucleotide immobilization or array formation using fluorous partitioning are extant either with or without direct MS detection, presumably due to the difficulties encountered in non-covalent immobilization and ionization of oligonucleotides.
Accordingly, it would be desirable to have materials, methods, and processes that do not suffer from one or more of the above drawbacks and would allow simultaneous assaying of a multitude of nucleotides with direct MS detection and facile analyte enrichment.